Based On FABP3 Mediated Mitochondrial Pathway, The Mechanism Of Yitangkang's Improvement On Myocardial Contractile Function In T2DM Rats Was Explored

Purpose:In this study,we investigated the correlation between fasting blood glucose(FPG),cholesterol(TC),fatty acid(TG),free fatty acid(FFA),myocardial FABP3 protein expression,myocardial ATP content,left ventricular ejection fraction(EF%),myocardial morphology,FABP3 protein expression and ATP content and EF% in vivo.The effect of Kang on FABP3 content and gene expression in myocardium of type 2 diabetic rats was studied,and its correlation was revealed.Then,the ATP content,respiratory chain complex(I,II),myocardial contraction-related protein expression and fatty acid metabolism-related protein expression in H9C2 myocardial cells were detected in vitro to observe whether Yitangkang could improve myocardial contraction energy by regulating the expression of mitochondrial mediators of FABP3.Thus,the mechanism is studied.Materials and Methods:Paper 1:SPF healthy male Westar rats weighing 200±20 g,2 months old,50 rats.Using random number table method,they were divided into 5 groups: normal control group(10),model control group(10),high dose group(10),middle dose group(10),and low dose group(10).The model control group,the high-dose group,the middle-dose group and the low-dose group were subjected to high-fat diet for 4 weeks,and then streptozotocin(STZ)45 mg/kg was intraperitoneally injected and replicated.Randomly selecting 4 model rats each to detect blood glucose above 16.7mmol / L was successful modeling.After the model control group,high-dose group,middle-dose group and low-dose group were successfully modeled,the normal control group and the model control group were given normal saline,and the high,middle and low groups were given different doses of traditional Chinese medicine Yitangkang.The decoction is administered by the stomach(the concentration of the decoction of the traditional Chinese medicine preparation is 100%,bottling and storage,and stored at 4 ° C).The clinical dose of Yitangkang is medium dose,2times is high dose,1/2 is low dose.The above method,once a day,for 4 weeks.The doseadministered is calculated according to the formula for conversion of human and biological crude drugs.Fasting blood glucose was measured at 2 and 4 weeks after the experimental intervention using Yitangkang as an intervention factor.After the end of treatment,the body weight,serum and myocardial tissue were weighed,and the fasting blood glucose(FPG),cholesterol(TC),fatty acid(TG)content,serum myocardial zymogram and free fatty acid(FFA),and myocardial contraction were measured.Diastolic function,myocardial morphology,expression of myocardial FABP3,ATP content in myocardium.The contents of fasting blood glucose(FPG),cholesterol(TC),fatty acid(TG)were detected by automatic biochemical analyzer,free fatty acid(FFA)was detected by Elisa kit,the expression of FABP3 protein in myocardium was detected by Western-blot,the content of ATP in myocardium was detected by colorimetry,and the systolic function of left ventricle was small.The myocardial morphology was observed by HE staining under light microscope.The correlation between FABP3 protein expression,ATP content and EF% was analyzed by SPSS software partial correlation analysis.Paper2:Sixty male SPF Wistar rats weighing(200 6550 Sixty rats were randomly divided into two groups,30 rats in the blank control group and 30 rats in the Yitangkang group.The rats in the blank control group were given distilled water twice a day.The rats in the blank control group were given 10% chloral hydrate anesthesia by intraperitoneal injection 2 hours after the 4th day of intragastric administration.The abdominal aorta was used for blood collection.The rats were left in the room for 2 hours.Heart(2500 r/min,4 C,20 min),then collected serum,56 C water bath inactivation for 30 min,after packaging,stored at-81 C.H9C2 cells were incubated in a DMEM incubator containing 10 mM glucose,10% FBS and 1% penicillin.The plasmid containing variable length FABP3 promoter was transfected and then administered with "Yi Tang Kang" serum.ATP content,respiratory chain complex(I,II),mitochondrial membrane potential(MMP)were detected by flow cytometry,myocardial contraction-related proteins(SERCA,RYR2,PLB)were detected by Western-blot,and fatty acid oxidation-related proteins(CPT-1,CPT-2)were detected by Western-blot.Results:Paper 1:1.The high-fat diet feeding combined with streptozotocin was used to prepare the type 2diabetic rat model,and the physiological indexes of each model rat were determined.Fasting blood glucose: 75% of fasting blood glucose >16.8mmol/L in each model rat(can guarantee that at least 10 rats in each group will continue the experiment),compared with the normal control group,the fasting blood glucose of each group is significantly increased(P<0.01);There was no significant difference between the high,middle and low groups in the model control group(P>0.05).Body weight: After modeling,compared with the normal control group,the body weight of each model group was significantly increased(P<0.01);there was no significant difference between the high,middle and low groups in the model control group(P>0.05).General conditions: rats in each model group showed polydipsia,polyphagia,and polyuria.2.Using Yitangkang as an intervention factor,the experimental intervention was carried out.The body weight and fasting blood glucose of each group were detected at 2 and 4 weeks after intervention.It was found that the body weight of each group was significantly lower than that of normal control group.The control group(P<0.05 or P<0.01);compared with the model control group,the high and middle dose groups were significantly increased(P<0.05),and there was no significant difference between the three dose groups(P>0.05).After 4 weeks of intervention,the body weight of each group was significantly lower than that of the normal control group(P<0.01).Compared with the model control group,the three dose groups were significantly increased(P<0.01).There was no significant difference between the dose groups(P>0.05).The fasting blood glucose level showed that compared with the normal control group,the fasting blood glucose of each group was significantly increased(P<0.05 or P<0.01);compared with the model control group,the fasting blood glucose was significantly lower in the high dose group(P< 0.05),there was no significant difference between the middle and low dose groups(P>0.05);there was no significant difference between the three dose groups(P>0.05).After 4 weeks of intervention,compared with the normal control group,the fasting blood glucose of each group was significantly higher(P<0.05 or P<0.01).Compared with themodel control group,the fasting blood glucose of the high and middle dose groups was significantly lower(P<0.01).There was no significant difference in the low-dose group(P>0.05);the low-dose group was significantly higher than the high-and medium-dose groups(P<0.01).3.The levels of TC,TG and FFA in the blood of the rats after the end of the experiment showed that compared with the normal control group,the TC and TG of the rats in each group were significantly increased(P<0.01).Compared with the model group,the three dose groups were compared.Both TC and TG were significantly lower(P<0.01);the highest dose was lower in the three dose groups(P<0.05 or P<0.01).4.4.After the end of the experiment,the cardiac function test showed that the ejection fraction of the model rats decreased significantly.Compared with the normal control group,the myocardial ATP content in the model control group decreased(p<0.01);the myocardial ATP content in the low dose group,the middle dose group and the high dose group was significantly higher than that in the model control group(p<0.01).5.The content of FABP3 protein in the myocardium of each group was detected after the end of the experiment.The protein expression and mRNA level of FABP3 in the model control group were significantly increased(P<0.01).The protein expression and mRNA levels of FABP3 in the low-dose,middle-dose,and high-dose groups were lower than those in the model control group and negatively correlated with the dose of Yitangkang,suggesting that the expression of FABP3 is increased in diabetic myocardial tissue.The expression of FABP3 decreased after treatment with Ginkang,and the inhibition of FABP3 expression increased with increasing dose.6.Controlling the expression of FABP3 protein,ATP content and EF% partial correlation coefficient = 0.791,P < 0.01,we can think that there is a linear relationship between ATP content and EF% belongs to a positive correlation.When ATP content was controlled,the partial correlation coefficient between FABP3 protein expression and EF% was-0.693,P <0.01,which suggested that there was a linear relationship between FABP3 protein expression and EF% and it was a negative correlation.Paper 2:1.Comparing the preparation effect of high,medium and low doses of Yitangkang serum,weshould use high doses of Yitangkang to prepare drug-containing serum for follow-up experiment.2.After transfection with FABP3 promoter plasmid with variable length and intervention with Yitangkang serum,ATP activity in H9c2 cells was detected by colorimetry.Compared with normal control group,ATP activity in cardiomyocytes of FABP3 overexpression group was significantly decreased(P < 0.01),and ATP activity in cardiomyocytes of drug-containing serum group was significantly decreased(P < 0.01).Compared with FABP3 overexpression group,ATP activity in cardiomyocytes of drug-containing serum group was significantly increased(P < 0.01).3.After transfection with FABP3 promoter plasmid containing variable length and intervention with Yitangkang serum,the results of colorimetric assay for respiratory chain complexes I and II in H9c2 cells showed that the contents of respiratory chain complexes I and II in myocardial cells of FABP3 overexpression group were significantly lower than those of normal control group(P < 0.01),while those of drug-containing serum were significantly lower than those of control group(P < 0.01).Respiratory chain complex I and respiratory chain complex II were significantly decreased(P < 0.01)and significantly increased(P < 0.05)in myocardial cells of the drug-containing serum group compared with the FABP3 overexpression group(P < 0.01).4.After transfection with FABP3 promoter plasmid containing variable length and intervention with Yitangkang serum,the content of myocardial contraction-related proteins(SERCA,RYR2,PLB)was detected by Western-blot.Compared with normal control group,the content of SERCA in myocardial cells of FABP3 overexpression group and drug-containing serum group decreased significantly.Compared with FABP3 overexpression group,SERCA content in cardiomyocytes of drug-containing serum group was significantly increased(P < 0.01).Compared with the normal control group,the content of RYR2 in cardiomyocytes of FABP3 overexpression group and drug-containing serum group decreased(P < 0.05),and the content of RYR2 in cardiomyocytes of drug-containing serum group was significantly higher than that of FABP3 overexpression group(P < 0.01).Compared with the normal control group,the content of PBL in myocardial cells of FABP3 overexpression group and drug-containing serum group increased significantly(P < 0.01),while that of FABP3overexpression group decreased significantly(P < 0.01).5.The content of fatty acid oxidation-related protein(CPT-1,CPT-2)in cardiomyocytes of FABP3 overexpression group was significantly lower than that of normal control group(P <0.01)after transfection with variable length FABP3 promoter plasmid and intervention with Yitangkang serum.Compared with FABP3 overexpression group,the content of CPT-1 in cardiomyocytes of drug serum group was significantly higher(P < 0.01).Compared with the normal control group,the content of CPT-2 in cardiomyocytes of FABP3 overexpression group and drug-containing serum group decreased significantly(P < 0.01),while the content of CPT-2 in cardiomyocytes of drug-containing serum group increased significantly(P <0.01).Conclusion:1.In vivo experiment,high-fat diet combined with intraperitoneal injection of streptozotocin can effectively prepare type 2 diabetic rat model,reduce the weight of the model rats,improve the fasting blood glucose level of the model rats,make them appear more than three or less symptoms,and in this experiment showed a success rate of 75%.Yitangkang can effectively reduce the level of fasting blood glucose in type 2 diabetic rats,effectively slow down the degree of weight loss in type 2 diabetic rats,effectively reduce the level of serum TC,TG,FFA in type 2 diabetic rats,significantly increase the ATP content in myocardial tissue,effectively inhibit the expression of FABP3 to improve myocardial contractility.The expression of FABP3 protein in myocardium of type 2 diabetic rats was negatively correlated with ejection fraction,and ATP was positively correlated with ejection fraction.2.In vitro,high-dose Yitangkang serum could significantly increase the ATP activity in FABP3-overexpressed cardiomyocytes and the contents of respiratory chain complexes I and II in the mitochondria of FABP3-overexpressed cardiomyocytes,suggesting that Yitangkang could promote the transport of electrons through complexes I and II.The activities of SERCA,RYR2 and PLB in FABP3-overexpressed cardiomyocytes were significantly increased,and the activities of CPT1 and CPT2 in FABP3-overexpressed cardiomyocytes were significantly increased.These results suggest that Yitangkang can improve the contractility of cardiomyocytes by improving the lipid metabolism-related proteins,the ATP productionenvironment and the level of ATP production in cardiomyocytes.